Increased epithelial permeability and oxidative stress-induced epithelial injury have been implicated in the pathogenesis of a wide spectrum of intestinal inflammatory diseases including necrotizing enterocolitis and inflammatory bowel disease (Crohn's disease and Ulcerative colitis). Our studies so far have shown that oxidative stress increases paracellular permeability in intestinal epithelial monolayers. The mechanism of this oxidative stress-induced disruption of epithelial junctions involves activation of a proto-oncogene, c-Src, tyrosine phosphorylation of junctional proteins, disruption of protein-protein interactions among the junctional proteins, and loss of integrity of the junctional complexes that prevent epithelial permeability. On the basis of our results it is further hypothesized that: a) oxidative stress-induced phosphorylation of occludin on specific tyrosine residues reduces its ability to bind ZO-1, ZO-2 and ZO-3, b) translocation of PP2A to the tight junction (TJ)plays a crucial role in oxidative stress-induced dephosphorylation of occludin on threonine residues and disruption of the tight junction, and c) EGF prevents oxidative stress-induced translocation of PP2A to the tight junction by PKCbeta1 and MAPK dependent mechanism. Using the above mentioned model we propose to determine that: 1) tyrosine phosphorylation of C-terminal tail of occludin reduces its ability to bind ZO-1, ZO-2 and ZO-3, 2) phosphorylation of occludin on specific tyrosine residue(s) is responsible for the prevention of its binding to ZO-1, ZO-2 and ZO-3, 3) mutation of specific tyrosine residue(s) on the C-terminal tail of occludin prevents the oxidative stress-induced disruption of tight junction, 4) oxidative stress induces translocation of PP2A to the tight junction by c-Src dependent mechanism, 5) expression of dominant negative PP2A-Cot and reduced expression by antisense oligos prevent oxidative stress-induced disruption of the tight junction, 6) dephosphorylation of occludin on serine/threonine attenuates its interaction with F-actin, 7) EGF prevents oxidative stress-induced translocation of PP2A to the T J, 8) PKCbeta1 mediates EGF-mediated protection of TJ, and 9) MEK and ERK are involved in the mechanism of EGF-mediated protection of TJ. Information derived from this study has the potential to expand our understanding of oxidative stress-mediated injury in intestinal epithelium, by identifying some of the mechanisms of oxidant-induced disruption of paracellular junctional complexes and protection by EGF.